Monoacetyldiacylglycerol derivative for the treatment of sepsis

ABSTRACT

The uses of mono acetyl diacyl glycerol derivatives extracted from deer antler for immunomodulating agent disclosed. Medical supplies and health foods containing the same as an effective ingredient also disclosed. Mono acetyl diacyl glycerol derivatives shows significantly effect for immuno modulation including immune enhancing. In the case of inducing cancer in a hamster by injecting cancer cell line, cancer development was delayed by activating lymphocytes, monocytes, and dendritic cells that are important factors to promote immunity and apoptosis of cancer cell was induced by promoting cytotoxicity of immune cell against cancer cell. Also in the case of mouse induced septic shock, it shows 100% survival rate even after lapse of 120 hours by control of immune function and suppression effect apoptosis. Therefore, mono acetyl diacyl glycerol derivatives according to the present invention can be effectively used for an immunomodulating agent, a sepsis treatment, a cancer treatment, and a health food for an immune modulation or the prevention of cancer.

BACKGROUND ART

This invention relates to uses of immunomodulating agent, medicalsupplies, and health foods containing mono acetyl diacyl glycerolderivatives extracted from deer antler as an effective ingredient.

Antler (in Latin, Cervi parvum cornu) is an uncalcified horn harvestedand dried from any animal of the deer family. In traditional orientalmedicine in Korea, deer antlers together with ginseng have been widelyused for their various acclaimed medicinal effects. The deer family forthe traditional use of antlers are limited only Cervus nippon Temminickvar. mantchuricus Swinhoe(Referred as C.N. hereafter) and Cervus elaphusL. Deer antler has been acclaimed to have numerous medicinal effects. Ithas been known to be efficacious in tonic agents, growth and developmentpromotion, hematopoiesis, treating nervous breakdown, treating cardiacinsufficiency, and generally improving the function of the five visceraand six entrails (Dong-euibogam, a classical medical literature inKorea). Other literatures in traditional medicine, concerning theeffects of deer antlers, also reported that tonic effects, nourishingeffect, strengthening vitality effects including improving cardiacfunction, relieving fatigue effects, enhancing immunity. Many attemptshave been made to uncover the curious chemical make-up of antler. As aresult, it is found to contain active gradients such as free aminoacids, trace (metallic) elements, hexose, pentose, hexosamine, uronicacid, sialic acid, mucopolysaccharides (e.g. hyaluronic acid,chondroitin A), various fatty acids, prostaglandins. It has also beenreported that glycolipid, phospholipid, cholesterol, hypoxanthine,cholest-5-ene-3β,7α-diol, cholesterol ester, polyamine were detected inthe extracts from deer antler. Others reported the presence of estrone,and estradiol receptor (report of NIH Korea, Vol. 22, p 359, 1985;Korean Biochem. J, Vol. 9, No. 3, p 153, 1976; Korean Biochem. J, Vol.9, No. 4; p 215, 1976; Korean Biochem. J, Vol. 10, No. 1, p 1, 1977;Shoykugaku Zasshi, 43(2), p 173, 1989).

Immunity is a defense mechanism protecting a living body from variouspathogens. Immunodeficiency is resulted from a defect in a constituentof immune system, indicating that immune system is unable to response tovarious antigens. Immunodeficiency is largely divided into congenital orprimary immunodeficiency and acquired or secondary immunodeficiency. Inthe case of congenital immunodeficiency, B-cells or T-cells do not existnaturally, so it can be treated only by gene therapy, antibody insertionand bone marrow transplantation. On the other hand, in the case ofacquired immunodeficiency, all the immune related factors existnaturally but there is malfunctioning in immune response, so it can beimproved by promoting the functions of immune factors. Recently theoutbreak of autoimmune diseases such as arthritis, atopy, dementia andsepsis have been increased. Autoimmune diseases are resulted from overincreasing of immune function. An immune suppressor has been used toremedy autoimmune diseases, but the immune suppressor also causesdecreasing of immunity frequently. Based on the disclosure of immunemechanism, various attempts have been made to develop an immuneregulator for the control of immunity. The purpose of these attempts isfor increasing defensive power of a living body against pathogens andminimizing side-effects by controlling promotion or suppression ofimmune function with immune regulators which can stimulate immune cellsnon-specifically. Immune regulators can remedy almost diseases of livingbody such as cancer, sepsis, degenerative arthritis, infection,dementia, aging, diabetes, anemia, skin disease, asthma, atopy, stress,nerve breakdown, physical fatigue, chronic fatigue syndrome, andosteoporosis. As of today, chemical compounds, microorganismcompositions, biological products, etc, have been used as an immuneregulator. Most of those immune regulators are limited in using becausethey are inclined to work only one effect (either immune promotion orsuppression). Therefore, they may cause side effects and have toxicitythemselves. In order to overcome above mentioned problems, foodstuffswithout toxicity, effective ingredients extracted from natural sourcesand the traditional herb medicines are the major targets to developimmune regulators and experiments to examine their effects as a medicinehave been on trial. But these immune regulators still have either immunepromotion or suppression effect.

Cancer, the leading cause of death in Korea, has been increasing everyyear. Chemo-therapy or radio-therapy for the treatment of cancer notonly kills cancer cells but also destroys normal bone marrow cells,especially hematopoietic cells regulating immunity and hematopoieses,resulting in the malfunction of immune system and hematopoietic organ(Korean J. BRM., 1, p 23, 1993; Korean J. BRM., 4, p 47, 1994; Crit. RevOncol Hematol. 1, p 227, 1984). Sepsis is a serious disease having over45% lethal rate caused by a severe systemic infection leading to asystemic inflammatory response. It almost happens when infected hostsresponse excessively against endotoxin from gram negative bacteria.However Antibiotics, steroid, or Xigris (Eli Lilly company) have beenused as a septic shock treatment, the lethal rate from septic shock isstill high because theses antibiotics, steroid, or Xigris areineffective against sepsis.

Thus, the present inventors separated various ingredients of C.N. antlerwhich has been known to having excellent pharmaceutical effects as afolk remedy, and further observed that one of those effectiveingredients of C.N. antler, mono acetyl diacyl glycerol, showedsignificant immune regulation activity in vivo. As a result of immuneregulation effects, the C.N. antler has a possibility of using forseptic shock treatment and anti-cancer agent without causing toxicity invivo. And, the present inventors completed this invention by confirmingthat mono acetyl diacyl glycerol of the invention can be used as a safeimmune enhancing agent, an immunomodulating agent, a septic shocktreatment and an anti-cancer agent.

DISCLOSURE Technical Problem

Therefore, it is an object of the present invention to provide animmunomodulating agent, a septic shock treatment, an anti-cancer agent,and health foods containing mono acetyl diacyl glycerol derivatives asan effective ingredient. Health foods are for modulating immune,preventing or treating septic shock and cancer.

Technical Solution

In order to achieve the above object, the present invention provides animmunomodulating agent containing mono acetyl diacyl glycerolderivatives represented by the following formula 1 as an effectiveingredient.

wherein, R1/R2 is 9-octadecenoyl(oleoyl)/hexadecanoyl(palmitoyl),hexadecanoyl (palmitoyl)/(9-octadecenoyl(oleoyl),hexadecanoyl(palmitoyl)/9,12-octadecadienoyl(linoleoyl),hexadecanoyl(palmitoyl)/9,12,15-octadecatrienoyl(linolenoyl) orhexadecanoyl(palmitoyl)/5,8,11,14-eicosatetraenoyl(arachidonoyl).

Here, above mentioned mono acetyl diacyl glycerol derivativesrepresented by the below formula 2 is preferred.

The present invention also provides an AIDS treatment, a sepsistreatment, and an anti-cancer agent containing mono acetyl diacylglycerol derivatives of formula 1 as an effective ingredient. Thepresent invention further provides health foods containing mono acetyldiacyl glycerol derivatives of formula 1 as an effective ingredient foran immune modulation or the prevention of cancer.

DESCRIPTION OF DRAWINGS

FIG. 1 is a set of photographs showing the T-cell (T-4 and T-8) activityof control group, IL-2 treated group (20 ng/ml), and Compound 3 treatedgroup (1 μg/ml). Each number indicates the number of spots capturingIL-2 specific antibody.

FIG. 2 is a graph showing the release of cytokines when T lymphocytesare activated by Compound 3,

{circle around (1)} Control group: anti-CD3, anti-CD28 treated,

{circle around (2)} Experimental group: anti-CD3, anti-CD28 and Compound3 (0.1, 1 μg/ml) treated.

FIG. 3 is a set of photographs showing the morphology of mouse dendriticcells derived from mouse bone marrow cells after the treatment of GM-CSF(20 ng/ml), IL-4 (20 ng/ml) and TNF-α (5 ng/ml).

A: a microscopic photograph taken right after the inoculation of mousebone marrow cells with the density of 1×10⁶ cells/ml (×100).

B: a microscopic photograph showing the round bone marrow stem cellsafter three days culture. Those cells formed a cluster, which wasgrowing on the bottom of a well of a cell culture plate (×400).

C: a microscopic photograph of the growing mature dendritic cells whichare forming cluster on the 6th day or 7th day of culture (×400), thesmall photograph is the enlarged photograph of the specific cell (×2).

D: a microscopic photograph of the dendritic cells which are formingspecific small and long protrusions on the 9th day of culture (×1000),the small photograph is the enlarged photograph of the specific cell(×2).

FIG. 4 is a set of graphs showing the results of FACS analyzing theexpressions of the dendritic cell specific markers, a monocyte specificmarker and a granulocyte specific marker on the 11th day of culture ofbone marrow cells separated from Balb/c AnN mouse. (Here, staining ofisomer control against hamster's IgG and rat's IgG2a is used for asetting marker line (straight line).

Following markers are used:

CD80 and CD86 as co-stimulation specific markers,

CD11c and DEC-205 as dendritic cell specific markers,

CD14 and F4/80 as monocyte/macrophage specific markers,

Gr-1 as a granulocyte specific marker.

FIG. 5 is an electrophoresis photograph showing the effects of Compound3 on dendritic cells on the expression of adhesion molecules.

Lane 1: Vcam-1 Lane 2: Icam-1 Lane 3: Icam-2 Lane 4: VLA-4 Lane 5: VLA-5Lane 6: LFA-1 Lane 7: GAPDH (+): Compound 3 treated group (−): Controlgroup.

FIG. 6 is a set of photographs showing the results of tumor forming atthe near injection site, 4 weeks after tumor (KIGB-5) i.v. injection andprocessing with different condition.

A: RPMI control group B: BMSC treated group C: BMSC + Ad/ΔE1 treatedgroup.

FIG. 7 is a set of photographs showing the results of tumor forming atthe near injection site, 8 weeks after tumor (KIGB-5) i.v. injection andprocessing with different condition.

A: RPMI control group

B: BMSC (2.5×10⁶ cells/day) treated group

C: BMSC (2.5×10⁶ cells/day)+Ad/ΔE1 (50 MOI) treated group

D: Dendritic Cells (5×10⁶ cells/day)+Tumor lysate treated group

E: BMSC (2.5×10⁶ cells/day)+Ad/IL-2 (50 MOI) treated group

F: Compound 3 (50 mg/kg/day) treated group.

FIGS. 8 and 9 are a set of photographs showing the gross and microscopicfindings of metastatic lung lesions of each group, 8 weeks after tumor(KIGB-5) i.v. injection.

In FIG. 8,

A: RPMI control group

B: BMSC (2.5×10⁶ cells/day) treated group

C: BMSC (2.5×10⁶ cells/day)+Ad/ΔE1 (50 MOI) treated group

D: BMSC (2.5×10⁶ cells/day)+Ad/IL-2 (50 MOI) treated group.

In FIG. 9,

A: RPMI control group

B: Dendritic Cells (5×10⁶ cells/day)+Tumor lysate treated group

C: Compound 3 (25 mg/kg/day) treated group.

FIG. 10 is a set of photographs showing the lung tumors and its size ofeach group, 12 weeks after tumor (KIGB-5) s.c (subcutaneously)injection.

A: RPMI control group

B: BMSC (2.5×10⁶ cells/day)+Ad/ΔE1 (50 MOI) treated group

C: BMSC (2.5×10⁶ cells/day) treated group

D: DC (5×10⁶ cells/day)+Tumor lysate treated group

E: BMSC (2.5×10⁶ cells/day)+Ad/IL-2 (50 MOI) treated group

F: BMSC (2.5×10⁶ cells/day)+Ad/IL-2 (50 MOI)+Compound 3 (25 mg/kg/day)treated group

G: Compound 3 (25 mg/kg/day) treated group.

FIG. 11 is a set of photographs showing gross findings of metastaticlung lesions of each group of Syrian golden hamsters treated withvarious doses of Compound 3, 8 weeks after biliary cancer cell ((5×10⁵cells) injection.

A: PBS Control group

B: Compound 3 (10 mg/kg/day) treated group

C: Compound 3 (25 mg/kg/day) treated group

D: Compound 3 (50 mg/kg/day) treated group

FIG. 12 is a set of photographs showing microscopic findings ofmetastatic lung lesions of each group of Syrian golden hamsters treatedwith various doses of Compound 3, 8 weeks after biliary cancer cell((5×10⁵ cells) injection.

A: PBS Control group

B: Compound 3 (10 mg/kg/day) treated group

C: Compound 3 (25 mg/kg/day) treated group

D: Compound 3 (50 mg/kg/day) treated group

FIG. 13 is a set of photographs showing gross findings of metastaticlung lesions of each of C57B1/6 mice received various treatments, 4weeks after melanoma cells (2×10⁴ cells) i.v. injection.

A: PBS Control group

B: Dendritic cells (4×10⁵ cells/day)+tumor lysate treated group

C: Compound 3 (50 mg/kg/day) treated group.

FIG. 14 is a set of photographs showing microscopic findings ofmetastatic lung lesions of each of C57B1/6 mice received varioustreatments, 4 weeks after melanoma cells (2×10⁴ cells) i.v. injection.

A: PBS Control group

B: Dendritic cells (4×10⁵ cells/day)+tumor lysate treated group

C: Compound 3 (50 mg/kg/day) treated group.

FIG. 15 is a graph showing survival rate of each treated group during 6weeks after melanoma cells (2×10⁴ cells) i.v. injection.

{circle around (1)} RPMI control group

{circle around (2)} Dendritic cells (5×10⁵ cells/day)+tumor lysatetreated group

{circle around (3)} Compound 3 (50 mg/kg/day) treated group.

FIG. 16 is a graph showing the cytotoxicity of T lymphocytes activatedby Compound 3 on melanoma cells

{circle around (1)} Control 1: anti-CD3, anti-CD28 treated group

{circle around (2)} Control 2: anti-CD3, anti-CD28, and IL-2 (20 ng/ml)treated group

{circle around (3)} Experimental group: anti-CD3, anti-CD28, andCompound 3 (1 μg/ml) treated group.

MODE FOR INVENTION

Hereinafter, the present invention is described in detail.

The present invention provides an immunomodulating agent, an AIDStreatment, a sepsis treatment, and an anti-cancer agent containing monoacetyl diacyl glycerol derivatives represented by the following formula1 as an effective ingredient. The compound represented by formula 1 ofthe present invention is one of following 5 compounds:

1)1-oleoyl-2-2palmitoyl-3-acetylglycerol(R1/R2=9-octadecenoyl/hexadecanoyl,referred as “Compound 1” hereinafter)

2)1-palmitoyl-2-oleoyl-3-acetylglycerol(R1/R2=hexadecanoyl/9-octadecenoyl,referred as “Compound 2” hereinafter)

3)1-palmitoyl-2-linoleyl-3-acetylglycerol(R1/R2=hexadecanoyl/9,12-octadecadienoyl, referred as “Compound 3” hereinafter)

4)1-palmitoyl-2-linolenoyl-3-acetylglycerol(R1/R2=hexadecanoyl/9,12,15-octadecatrienoyl,referred as “Compound 4” hereinafter)

5)1-palmitoyl-2-arachidonoyl-3-acetylglycerol(R1/R2=hexadecanoyl/5,8,11,14-eicosatetraenoyl, referred as “Compound 5” hereinafter)

Any of mono acetyl diacyl glycerol derivative represented by formula 2is available and in particular, Compound 3 is preferred. The compound ofthe present invention was extracted from C.N. antlers or manufactured byconventional organic synthesis method. The exemplary of extractionmethod has the following steps. Particularly, the chloroform extracts ofC.N. antler are obtained by extracting C.N. antler with hexane first andfurther extracting the residue of the hexane extract with chloroform.The amounts of hexane and chloroform used in this extraction process areenough amounts to impregnate deer antler. Generally, hexane andchloroform are used in the ratio of 4˜5 l to 1 kg of C.N. antler. Thechloroform extract of C.N. antler obtained from such extractionprocesses, is fractionated and purified by a series of silica gel columnchromatography and TLC (Thin layer chromatography). An eluent of thesubsequent extracting steps is selected from chloroform/methanol,hexane/ethylacetate. In order to synthesize mono acetyl diacyl glycerolderivatives chemically, for instance, 1-palmitoylglycerine is separatedfrom the products in the reaction of both glycerol and palmitic acid.The objecting mono acetyl diacyl glycerol can be synthesized asesterifying 1-palmitoylglycerine with carboxylic acid compounds such asacetic acid and linoleic acid, and purified as occasion demands. Anothermethod for synthesizing mono acetyl diacyl glycerol derivatives is theacetolysis of phosphatidyl choline.

The mono acetyl diacyl glycerol compound according to the presentinvention is for immunomodulating agent. Immunity modulation includesincreasing deteriorated immunity abnormally or maintaining the balanceof increased immunity abnormally. Therefore, mono acetyl diacyl glycerolcompounds according to the present invention have effects of not onlypreventing and treating various diseases resulted from deterioratedimmune system and caner but also inhibiting, preventing, and treatingautoimmune diseases such as arthritis, atopy, dementia, and sepsisresulted from autoimmune reaction.

In the regulation of immune function, the important thing is notincrease of T cell which is responsible for immunity but the extent of Tcell's activation, the ratio of T4 to T8 cells, and the kinds ofcytokines secreted from T4 and T8 cells. The present inventors treatedmono acetyl diacyl glycerol derivatives to T-4 and T-8 lymphocytes forresearching the immunomodulating effect of mono acetyl diacyl glycerolderivatives of the invention. As a result, it was confirmed thatsecretion of IL-2, a kind of cytokines, was increased in those cells(see FIG. 1). After treating the cells with the Compound 3 of thepresent invention by using Bio-plex, which enables measuring huge amountof cytokines at a time, the secretion of cytokine in T-cells wasinvestigated. As a result, the secretions of IL-2, IL-4, and IL-5 weremuch greater in Compound 3 treated group than in a control group (seeFIG. 2). The most increased cytokine, IL-4 is a multi-function cytokinecalled anti-inflammation cytokine which is secreted from Th2 which isdifferentiated from T4 cells. As inhibiting differentiation of T4 toTh1, IL-4 can suppress cell damage resulted from autoimmune reaction byprocessing important role to anti-cancer effect and immune responseregulation (Annu. Rev. Immunol. 1999. 17: 701˜738). The compounds of thepresent invention have effects of both immunity enhancing by stimulatingIL-2 secretion and immunity modulating by stimulating IL-4 secretion.And, the compounds of the present invention also can maintain the ratioof T4 to T8 normally by increasing and activating not only T4 but alsoT8, which is a cytotoxic immune cell. Therefore, it is effective fortreating on side effects and diseases resulted from abnormal increasingor decreasing of immune system. In the septic shock model, theseimmunity enhancing effects can work to the direction of stimulating IL-4secretion and inhibiting apoptosis. In result, the lethal rate of sepsisis decreased remarkably. Therefore, mono acetyl diacyl glycerolderivatives according to the present invention are useful for thetreatment of autoimmune diseases, for instance the preventing andtreating of sepsis, because these compounds increase IL-4 secretion.

It has been known to that the interaction between cells stimulatevarious hematopoietic cells and immune cells, and particularly,dendritic cells are very important in immune system. The presentinventors investigated the effect of mono acetyl diacyl glycerolderivatives on the interaction between separated and induced dendriticcells and TCR (T-cell Receptor). For the investigation, RT-PCR of theCompound 3 treated DC (Dendritic Cells) was performed to measure theexpressions of adhesion molecules mediating the interaction between DCand TCR. As a result, the expression of adhesive molecules such asVcam-1, Icam-1, Icam-2, VLA-4, VLA-5, and LFA-1 were increased,comparing to a control (see FIG. 5). From the above results, mono acetyldiacyl glycerol derivatives according to the present invention wereconfirmed to have the effects not only T-cell activation effect but alsospecific anti-cancer effect through activating of dendritic cells whichenable T-cell to recognize antigen of cancer cells.

From the above results, mono acetyl diacyl glycerol derivativesaccording to the present invention were confirmed to have the immunityenhancing effect by increasing cytokine secretion through activatingT-cells and by promoting the proliferation and stimulation ofhematopoietic cells and immune cells through increasing the expressionsof intracellular adhesion molecules. As a result, it was confirmed thatthese compounds have the possibility of using as an immuno-therapyagainst various diseases. For instance, mono acetyl diacyl glycerolderivatives according to the present invention can be used as treatmentor health food for enhancing immunity in human AIDS patients by theproliferation effect of T4 and T8 cells. In the early phase of the AIDSpatients, T4 was decreased but serious outbreak did not happen. On theother hand, in the late phase of the AIDS patient, T8 was decreased andserious outbreak happened. Therefore, the ratio of T4 to T8 is animportant factor and the absolute number of T4 and T8 is also animportant factor. Further, the present inventors confirmed that themodulation of immune function by the increasing of the IL-4 secretion iseffective for various autoimmune diseases. In order to investigate theuse of compounds according to the present invention as prevention andtreatment for septic shock, the CLP (Cecal Ligation and Puncture) testof mice was performed. In result, all tested mice survived until 120hours. Therefore, it confirmed that mono acetyl diacyl glycerolderivatives according to the present invention were effective forpreventing and treating of sepsis. From the above results, it confirmedthat mono acetyl diacyl glycerol derivatives according to the presentinvention were good for ideal immunomodulating agent having bothimmunity enhancing effect and immunity function regulation effect.

Further, in order to investigate the use of compounds according to thepresent invention for prevention and treatment of cancer, the presentinventors investigate anti-cancer effect of the compounds againstbiliary cancer and malignant melanoma that were known to the incurablecancer. First, the present inventors induced cancer in a hamster byinjecting intravenously or subcutaneously KIBG-5, a biliary cancer cellline. Then, RPMI, BMSC, adenovirus/ΔE1, dendritic cell+tumor lysate,Compound 3, adenovirus/IL-2 and the mixtures were injected to a hamster.The observation of result was performed 4 weeks later. As a result, whenit observed by the naked eye or a microscope, dendritic cell+tumorlysate, Compound 3, and adenovirus/IL-2 treated groups did not formtumor (see FIG. 6). Further, tumor cells were injected intravenously andobservation was performed 8 weeks later. As a result, metastatic lunglesion was formed in all groups except BMSC+adenovirus/IL-2 treatedgroup. From the biopsy, only a minute lesion was observed in dendriticcell+tumor lysate treated group and Compound 3 treated group (see FIG.7, FIG. 8, and FIG. 9). And further, tumor cells were injectedsubcutaneously and observation was performed 12 weeks later. As aresult, tumor was formed in all groups except BMSC+adenovirus/hIL-2treated group and BMSC+Ad/hIL-2+Compound 3 treated group (see FIG. 10).The tumor formation was inhibited by Compound 3 dose-dependently (seeFIGS. 11 and 12). As explained hereinbefore, the present inventorsinduced metastatic cancer in hamster by injecting biliary cancer cells(KIBG), and then treated the hamster with mono acetyl diacyl glycerolderivatives of the present invention. As a result, it was confirm thatcancer development was significantly inhibited by the treatment of thosecompounds of the present invention.

Intravenous injection of malignant melanoma cells was performed to thetail of mice to induce cancer therein. Then, each of or the mixture ofRPMI, dendritic cells (DC), tumor lysate and Compound 3 was treated. Asa result, metastatic lung lesion was formed in a control group treatedwith RPMI, but no lesions were observed in the groups each treated withCompound 3 and dendritic cells+tumor lysate (see FIGS. 13 and 14). Inaddition, Compound 3 treated group and dendritic cells+tumor lysatetreated group were observed for 6 weeks after tumor injection, resultingin 90% survival rate (see FIG. 15). Based on the above results, thepresent inventors confirmed that Compound 3 activates T-cell (T4 and 8),which means it has anti-cancer effect. So, the present inventorsperformed cytotoxicity test of T-cells activated by Compound 3 tomalignant melanoma in vitro. As a result, cytotoxicity was increasedmuch when T-cells were treated with Compound 3 than when T-cells werenot treated with Compound 3, and also cytotoxicity was increased withthe increase of the amount of T-cells (see FIG. 16). As explainedhereinbefore, it was confirmed that mono acetyl diacyl glycerolderivatives of the present invention inhibit cancer development and showcytotoxicity to cancer cells by activating T-cells, indicating that thecompounds of the present invention can be effectively used as ananti-cancer agent. The treatment with the product of the presentinvention as an anti-cancer agent appears to be promising for bile ductcancer, kidney cancer and melanoma, but other forms of malignantdiseases should be explored.

The present inventor, henceforth, completed this invention by preparingtrial capsules and tablets containing mono acetyl diacyl glycerolderivatives as an effective ingredient. An immunomodulating agent, anAIDS treatment, a sepsis treatment, and an anti-cancer agent of thepresent invention preferably include mono acetyl diacyl glycerolderivatives by 20 to 100 weight % to the total weight of compounds, morepreferably include them by 30 to 100 weight %. If the amount of monoacetyl diacyl glycerols is too much or less, it just difficult to takemedicine and there are no advantages. It is also preferred for a sepsistreatment, an anti-cancer agent, and an immunomodulating agent to beorally administered 1 to 3 times/day or 1 to 4 times/day with the doseof 50 mg/kg. The compounds according to the present invention canadditionally include one or more pharmaceutically acceptable carriers,in addition to an effective ingredient, to be formulated in apharmaceutical form. The carrier can be selected from a group consistingof saline, buffered saline, water, glycerol and ethanol, but theselection is not always limited thereto. Any acceptable pharmaceuticalformulation know in this field (Remingtons Pharmaceutical Science (thelatest edition), Mack Publishing Company, Easton Pa.) is available. Acomposition of the present invention can be administered orally and beused in general forms of pharmaceutical formulations. The composition ofthe present invention can be prepared for oral administration by mixingwith generally used fillers, extenders, binders, wetting agents,disintegrating agents, diluents such as surfactant, or excipient. Theeffective dosage of the composition of the present invention can bedetermined according to age, gender, health condition, absorption of anactive ingredient, inactivation rate, excretion and other medicinesapplied together. For example, the dosage for oral administration mightbe 0.24 to 9.0 g per day, but not always limited thereto. The presentinvention also includes pharmaceutical formulations in dosage units.This means that the formulations are presented in the form of individualparts, for example tablets, coated tablets, capsules, pills,suppositories and ampoules, the active compound content of whichcorresponds to a fraction or a multiple of an individual dose. Thedosage units can contain, for example, 1, 2, 3, or 4 individual doses or½, ⅓, or ¼ of an individual dose. An individual dose preferably containsthe amount of active compound which is administered in one applicationand which usually corresponds to a whole, ½, ⅓ or ¼ of a daily dose.Solid formulations for oral administration are tablets, pills, dustingpowders and capsules, liquid formulations for oral administration aresuspensions, solutions, emulsions and syrups, and the above mentionedformulations can contain various excipients such as wetting agents,sweeteners, aromatics and preservatives in addition to generally-usedsimple diluents such as water and liquid paraffin. The compounds of thepresent invention can be applied not only formulations for oraladministration but also formulation for injection. For example, wateryor oily suspension for sterile injection can be prepared according tothe known method with dispersing agents, wetting agents or emulsions.Any acceptable pharmaceutical solvent includes water, Ringer's solution,or isotonic NaCl solution. Sterile fixing oil is used as solvent ordispersive medium and can include non-stimulus fixing oil includingmonoglyceride, diglyceride, and poly propylene glycol and fatty acidsuch as oleic acid.

The present invention also provides immunomodulating and anti-cancerhealth food containing mono acetyl diacyl glycerol derivatives as aneffective ingredient. In the present invention, “health food” includesfoodstuff, nutrient and health supplement for treating or preventing ofvarious diseases and maintaining the balance of body function. Healthfood prepared in the present invention contains mono acetyl diacylglycerols by 0.02 to 100 weight %. In the case of using the compounds ofthe present invention as health food, the compounds can be usedaccording to the conventional method, for example, using intactcompounds or using mixed compounds with other foods or food ingredients.The effective amount of the compound mixture depends on the purpose ofits use (prevention, health or therapeutic treatment). In the case ofusing for prevention, the preferable amount of mono acetyl diacylglycerol derivatives is from 0.02 to 2 weight % for the total amount ofhealth food, preferably 0.2 to 0.6 weight %. If the amount of monoacetyl diacyl glycerols is too much or less, it just difficult to takehealth food and there are no advantages. The effective ingredient isalso safe for the long-term administration aiming at the control or thepreservation of health, supported by cytotoxicity test. Any kinds offood containing the composition of the present invention can be madewithout limitation. For example, meat sausage, bread, soups, beverages,teas, drinks, alcoholic beverages and vitamin complex are the food to bemade as health food containing the composition of the present invention.In case that the health food is used as nutrients or health supplementsfor the purpose of treating and preventing disease, the preferableamount of mono acetyl diacyl glycerol derivatives is from 20 to 100weight % for the total amount of health food, preferably 30 to 100weight %, more preferably 35 to 95 weight. The intake might be 0.18 to9.0 g per day, but not always limited thereto. The formulations includetablets and capsules.

As explained hereinbefore, mono acetyl diacyl glycerol derivatives ofthe present invention activate T-cells to promote the secretion ofcytokines, increase the expression of adhesive molecules between cellsto stimulate hematopoietic cells and immune cells so as to not onlyimprove immunity but also prevent and treat autoimmune disease andcancer.

Hereinafter, the preferable experimental examples are provided forbetter understanding of the present invention. However, the presentinvention is not limited to the following experimental examples.

EXPERIMENTAL EXAMPLE 1 Effects of Mono Acetyl Diacyl GlycerolDerivatives on T-Cell and Mononuclear Cell Proliferation EXPERIMENTALEXAMPLE 1-1 Effects of Mono Acetyl Diacyl Glycerol Derivatives on T-CellProliferation

Splenocytes were collected from C57BL/6 mice (provided from AsanInstitute for Life Sciences Animal Lab., Seoul, Korea) spleens. Then,single cell suspensions were obtained by repeated aspiration andflushing. Red blood cells were removed using ammonium chloride and thenpassed through nylon wool to remove debris and clumps. T-cells werepurified using magnetic bead (MACS bead, Miltenyi Biotec, bergichgladbach, Germany) containing anti-goat IgG MACS bead, Miltenyi Biotec,bergich gladbach, Germany) or anti-mouse CD4 (MACS bead, MiltenyiBiotec, bergich gladbach, Germany) or anti-mouse CD8 antibody (MACSbead, Miltenyi Biotec, bergich gladbach, Germany) (Turner and Dockrell(1996) Immunology, 87: 339-342).

T cell suspensions were suspended in Isocove's modified Dulbecco'smedium (IMDM, Gibco, Grand Island, N.Y.) supplemented with 10% fetalbovine serum (referred as ‘FBS’ hereinafter) (Gibco, Grand Island,N.Y.). 5×10⁴ viable cells per well were cultured in 96-well plates, with1 μg/ml, of Compound 1, Compound 2, Compound 4 and Compound 5, 0.01,0.1, 1 μg/ml of Compound 3 (synthesized and provided by Department ofChemistry, Ewha Womans University, Seoul, Korea) or 20 ng/ml of IL-2. Onthe 6th day, cells were incubated with 1 μCi ³H-thymidine/well for 24hours. On the 7^(th) day, the cells were harvested and the incorporationIndex (referred as ‘SI’ hereinafter) was calculated by the followingMathematical Formula 1.SI= ³H-thymidine absorbed by wells of experimental group (CPM insample)/³H-thymidine absorbed by wells of control group (CPM incontrol)  [Mathematical Formula 1]

As a result, mono acetyl diacyl glycerol derivatives treated group hadincreased Si of T-cells by thymidine uptake of 2.05, which was similarto that of IL-2 treated group (Table 1).

TABLE 1 Treated group SI IL-2 (20 ng/ml)* 2.05 ± 0.24 Compound 1 (1μg/ml)* 2.01 ± 0.43 Compound 2 (1 μg/ml)* 2.03 ± 0.54 Compound 3 (0.01μg/ml)** 1.83 ± 0.32 Compound 3 (0.1 μg/ml)* 1.96 ± 0.18 Compound 3 (1μg/ml)* 2.05 ± 0.64 Compound 4 (1 μg/ml)* 1.98 ± 0.26 Compound 5 (1μg/ml)* 2.02 ± 0.38 *P < 0.05, **P < 0.005. All tests were done intriplicate and were repeated three times.

EXPERIMENTAL EXAMPLE 1-2 Effects of Mono Acetyl Diacyl GlycerolDerivatives on Monocytes Proliferation

Monocytes were isolated from human whole blood using Histopaque 1077.And then, monocytes (5×10⁶ cells/ml) were allowed to adhere to tissueculture flask for 3 hours in a 5% CO₂ incubator. After 3 hours,non-adherent cells were removed and adherent cells were placed in96-well plates in RPMI 1640 medium (GIBCO, Grand Island, N.Y.)supplemented with 10% FBS. 5×10⁴ viable cells per well were cultured in96-well plates, with 1 μg/ml of Compound 1˜5. On the 6^(th) day, cellswere incubated with 1 μCi ³H-thymidine/well for 24 hours. On the 7^(th)day, the cells were harvested and the incorporation of ³H-thymidine wasmeasured. Si was calculated by the above mentioned formula 1. As aresult, Compound 1-5 treated group had increased monocytes SI 10.68,comparing to control, indicating that the compounds stimulatedproliferation of monocytes (Table 2).

TABLE 2 Treated group SI (±S.E) Non treated control 1 Compound 1 (1μg/ml)*  9.97 ± 0.10 Compound 2 (1 μg/ml)* 10.42 ± 0.15 Compound 3 (1μg/ml)* 10.68 ± 0.13 Compound 4 (1 μg/ml)* 10.21 ± 0.18 Compound 5 (1μg/ml)*  9.75 ± 0.09 *P < 0.001, All tests were done in triplicate andwere repeated two times.

EXPERIMENTAL EXAMPLE 2 Effects of Compound 3 on T Cell ActivityEXPERIMENTAL EXAMPLE 2-1 Measurement of Cytokine by Elispot

Elispot bioassay (ESAT-6 enzyme-linked immunospot assay) is a verysensitive quantification assay for detecting cytokine bound to themembrane because the bottom of each well of Elispot plates used in thisassay was pre-coated with a cytokine specific antibody. Thus, Elispotassay was performed to measure the T cell activity. T-cells were seededby 2×10⁶ cells to each well in a 24-well sterile tissue culture plate(Nunc, Denmark), followed by the treatment with 0.01, 0.1, 1 μg/mlconcentrations of Compound 3 or IL-2 (20 ng/ml). On the 7^(th) day,cells were harvested and the cells were seeded by 5×10⁵ cells/ml inmulti-testplates (Elispot system kit, AID, Straberg, Germany) coatedwith the respective primary antibody (murine IL-2). After the plate wasincubated for 24 hours in a 5% CO₂ incubator, there was a secretion ofcytokines by the cells, which were captured by the primary antibody(murine IL-2) determined by Elispot using commercially available mouseIL-2 Elispot kits according to the manufacturer's instructions. Eachsample was tested in duplicate. Counting the number of IL-2 producingcells by Elispot is accomplished with Elispot reader (AID Elispot ReaderSystem). The results showed that Compound 3 treated group showed 1.52folds increased T-4 activity, comparing to control group, and 1.46 foldsincreased of T-8 activity (FIG. 1).

EXPERIMENTAL EXAMPLE 2-2 Measurement of Cytokine by Bio-Plex

Bio-plex can measure huge amount of cytokine at a time in a well. Thus,Bio-plex kit was used to quantify 8 kinds of cytokines of Th1/Th2channels, which are secreted when T-cells are activated. Sterilized24-well tissue culture plate (Nunc, Denmark) was treated with anti-CD3and anti-CD28. Then, the plate was inoculated with T-cells by 2×10⁶cells/ml. In order to activate T-cells, 0.1, 1 μg/ml of Compound 3 wastreated thereto, followed by culture for 5 days. On the 5^(th) day,culture solutions at each different stage were recovered, followed bycentrifugation. Supernatants were obtained and cytokine secreted thereinwas quantified by using Bio-plex kit according to the manufacturer'sinstruction (Bio-rad). As a result, three kinds of cytokines (IL-2,IL-4, and IL-5), among 8 kinds of cytokines (IL-2, IL-4, IL-5, IL-10,IL-12, INF-γ, GM-CSF, TNF-α) were secreted in the group treated withCompound 3 and the amounts of them were bigger than those in a controlgroup not treated with Compound 3 (FIG. 2).

EXPERIMENTAL EXAMPLE 3 T Cell Proliferation Assay

The following experiment was performed to confirm the effect of Compound3 against immunocytes of the AIDS patients. First, Human mononuclearcells were obtained by Hisopaque 1077 from peripheral blood of AIDSpatients. Red blood cells were removed using ammonium chloride and thenpassed through nylon wool to remove debris and clumps. T-cells werepurified using magnetic bead (anti-human CD3) (MACS bead, MiltenyiBiotec, bergich gladbach, Germany). T cell suspensions were suspended inIsocove's modified Dulbecco's medium (IMDM, Gibco, Grand Island, N.Y.)supplemented with 10% fetal bovine serum (referred as ‘FBS’ hereinafter)(Gibco, Grand Island, N.Y.). 5×10⁴ viable cells per well (in triplicate)were cultured in 96-well plates, with 0.01, 0.1, 1 μg/ml concentrationsof Compound 3 or IL-2 (20 ng/ml). On the 6th day, cells were incubatedwith 1 μCi ³H-thymidine/well for 24 hours. On the 7^(th) day, the cellswere harvested and the incorporation of ³H-thymidine was measured. TheSI (Stimulation Index) was calculated by the above mentioned formula 1.As a result, in AIDS patients, T cell proliferation assay, showedCompounds 3 treated group had 1.5 to 3.9 fold increase of T-cellstimulation index by thymidine uptake in all patients (4 out of 4)compared with control as seen in Table 3. Over all result of stimulationby Compound 3 was comparable with IL-2 stimulation.

TABLE 3 Stimulation Index (SI) 1 2 3 4 IL-2(20 ng/ml) 1.41 4.17 1.296.54 Compound 3 (1 μg/ml) 2.23 3.87 1.48 3.49

EXPERIMENTAL EXAMPLE 4 Effects of Compound 3 on the Expression ofAdhesion Molecules of Dendritic Cells EXPERIMENTAL EXAMPLE 4-1 DendriticCell Culture

Bone marrow cells were obtained from the femurs and tibias of Balb/c AnNmice (Park, J. et al. (2003) J. Korean Med. Sci., 18: 372-380). Thecells were washed 3 times in RPMI, and then mononuclear cells wereobtained. These mononuclear cells were allowed to adhere to tissueculture flask 3 hours in RPMI and 10% FBS. After incubation, theadherent cells (monocytes) were removed and non-adherent cells wereplaced in 100 mm tissue culture dishes, in a concentration of 1×10⁵cells/ml in RPMI plus 10% FBS supplemented with 20 ng/ml murine rGM-CSF(R & D systems, Minneapolis, Minn., USA), 10 ng/ml, murine IL-4 (R&Dsystems), and 2.5 ng/ml murine TNF-α (R & D systems). Culture disheswere fed every 3 days. Murine TNF-α (R & D systems) was added at the6^(th) day of the culture. After that, murine TNF-α (R & D systems) wasadded every 3 days until on the 11th day. Mature dendritic cells wereharvested for RT-PCR of adhesion molecule studies. As a result, whenround shaped granulocytes were cultured for three days, those cellsformed a cluster which was growing on the bottom of a well of cellculture plate, and mature dendritic cells were growing with forming agroup on the 6^(th) or the 7^(th) day of culture. And on the 9^(th) dayof culture, dendritic cells formed a small but long protrusionspecifically (FIG. 3).

EXPERIMENTAL EXAMPLE 4-2 Determination of Dendritic Cell Phenotype

Those cells that were big and negative against trypan blue staining werecounted and the morphology of each of them was investigated. 1×10⁶cells/ml were cultured and then washed, followed by fixation with 1%para-formaldehyde solution. Flow cytometric analysis of the fixed cellswas performed by using FACScan (Beckton Dickinson, Mountain View,Calif., U.S.A), leading to the determination of the phenotype usingantibodies against the following markers; isotype control againsthamster IgG, rat IgG 2a, DC marker: DEC 205 (NLDC-145) and CD 11C,co-stimulatory/adhesion molecule: CD 80 (B7-1) and CD 86 (B7-2),macrophage marker: CD 14 and F4/80, granulocyte marker: Gr-1(Pharmingen, Hamburg, Germany). As a result, the levels ofco-stimulation specific molecular markers CD80 and C86 and dendriticcell specific markers CD11C and DEC-205 were high. On the contrary, thelevels of monocytes specific markers CD14 and F4/80 and granulocytespecific marker Gr-1 were low. The results indicate that the dendriticcells separated in the present invention have an exact phenotype ofdendritic cells and the purity 97 to 98% (FIG. 4).

EXPERIMENTAL EXAMPLE 4-3 Treatment and the Expression of AdhesionMolecules

It is generally known that the cell-cell interaction is involved instimulations of various hematopoietic cells and of immune cells. Thus,the present inventors tried to confirm whether or not Compound 3 affectsvarious adhesion molecules of the mentioned cells. Particularly,dendritic cells cultured in the above example were treated with 1 μg/mlof Compound 3, and then RT-PCR was performed.

Following primers: Icam-1 (SEQ. ID. No 1 and No 2), Icam-2 (SEQ. ID. No3 and No 4), Vcam-1 (SEQ. ID. No 5 and No 6), VLA-4 (SEQ. ID. No 7 andNo 8), VLA-5 (SEQ. ID. No 9 and No 10), LFA-1 (SEQ. ID. No 11 and No 12)and GAPDH (SEQ. ID. No 13 and No 14) were used for the RT-PCR. Reactionsets used herein was a mixed solution of 2 μl DNA, 10× buffer solution,1.5 μl of MgCl₂, 2 μl of dNTPs, 0.5 μl of forward primer, 0.5 μl ofreverse primer, 0.2 μl of polymerase and 15.8 μl of distilled water.

Total RNA separated from dendritic cells and MS-5, low density cellscultured with oligo(dt)-primer, was reverse-transcribed, and PCR wasperformed at 94° C. for 30 seconds, 65° C. for 30 seconds and 72° C. for50 seconds. PCR was performed 34 times at total, and PCR products weredoubled every performance. The expressions of adhesion molecules such asVcam-1, Icam-1, Icam-2, VLA-4, VLA-5, and LFA-1 were confirmed byRT-PCR. For the quantification, PCR with GAPDH was performed to confirmthe corresponding cDNA. The results showed that the expressions ofadhesion molecules, Icam-2, VLA-5, LFA-1 on Compound 3 treated dendriticcells were significantly increased compared to a control (FIG. 5).

EXPERIMENTAL EXAMPLE 5 Study on Anti-Cancer Effect of the Compound 3through Subcutaneous Injection (Local Model) and Intravenous Injection(Systemic Model) EXPERIMENTAL EXAMPLE 5-1 Biliary Cancer Model inHamster

Six week old female Syrian golden hamsters (Harlan, Indianapolis, India,USA) were housed in specific pathogen free unit. 5×10⁵ KIBG-5 cells(Molecular therapy, Vol. 3, No. 4, pp 431-437) suspended in 100 μl ofRPMI 1640 serum-free medium were intravenously injected via femoralvein. And 5×10⁵ KIBG-5 cells suspended in 100 μl of RPMI 1640 serum-freemedium were subcutaneously injected to the flank of the hamsters.Hamsters injected KIBG-5 were divided into following 7 groups;

1) Control group treated with RPMI medium,

2) Experimental group treated with non-modified BMSC cells (2.5×10⁶)(Leukemia & Lymphoma, Vol. 44, No. 11, pp 1973-1978),

3) Experimental group treated with BMSC cells modified with Ad/ΔE1 50MOI (Leukemia & Lymphoma, Vol. 44, No. 11, pp 1973-1978),

4) Experimental group treated with DC+ tumor lysate (5×10⁶),

5) Experimental group treated with BMSC cells modified with Ad/hIL-2 50MOI (Leukemia & Lymphoma, Vol. 44, No. 11, pp 1973-1978),

6) Experimental group treated with BMSC cells modified with Ad/hIL-2 50MOI+Experimental group treated with Compound 3 (25 mg/kg/day),

7) Experimental group treated with Compound 3 (25 mg/kg/day).

One week after the injection of cancer cells to hamsters (BMSC treatedgroup), 2.5×10⁶ of BMSC cells were injected once again to each hamster.In the case of DC+tumor lysate treated group, 5×10⁶ of DC cells andtumor lysate were injected to each hamster (with subcutaneous injectionor intravenous injection) at the first, second, third, forth, sixth,eighth week, followed by observation for 12 weeks. In the case ofCompound 3 treated group, one week before KIBG-5 cells injection,Compound 3 (25 mg/kg/day) via P.O was continued 2 weeks on and 1 weekoff for 8 weeks.

As a result, 4 weeks after the cancer cell injection, tumor formed inRPMI treated group which was a control, BMSC treated group, andBMSC+Ad/ΔE1 treated group, but no tumor were found in DC+tumor lysatetreated group, Compound 3 treated group, and BMSC+Ad/IL-2 treated group(FIG. 6). Moreover, 8 weeks after the cancer cell injection, multiplemetastatic lung lesions were found in RPMI treated group which was acontrol, BMSC treated group, and BMSC+Ad/ΔE1 treated group, and only oneminute lung lesion was found in DC+ tumor lysate treated group, andCompound 3 treated group. But no lesions were found in BMSC+Ad/IL-2treated group (FIGS. 7, 8, 9). Further, 12 weeks after the cancer cellsubcutaneous injection, tumor formed in RPMI treated group which was acontrol, BMSC treated group, BMSC+Ad/ΔE1 treated group, and DC+tumorlysate treated group, and less 5 mm sized tumor in one mice were foundin BMSC+Ad/hIL-2 treated group but no tumor were found inBMSC+Ad/IL-2+Compound 3 treated group (FIG. 10).

In another hand, six week old female Syrian golden hamsters were housedin specific pathogen free unit. KIGB-5 cells (5×10⁵) suspended in 100 μlof RPMI 1640 serum-free medium were intravenously injected via femoralvein. Hamsters were divided into following 4 groups: 1) PBS controlgroup, 2) Compound 3 (10 mg/kg/day) treated group, 3) Compound 3 (25mg/kg/day) treated group, 4) Compound 3 (50 mg/kg/day) treated group.One week before tumor cell injection, Compound 3 (10, 25 or 50mg/kg/day) via P.O was continued 2 weeks on and 1 week off for 12 weeks.Animals of each group were sacrificed at 4, 8, 12 weeks for pathologicalexamination. Gross findings at 4^(th) week, tumor developed at injectionsite in control group, Compound 3 (10, 25 or 50 mg/kg/day) treatedgroups showed no evidence of tumor. At 8^(th) week, control groupobserved multiple metastatic lesions in both lungs. Compound 3 treatedgroup (25, 50 mg/kg/day) did not show any metastatic lung lesions withnaked eye, but Compound 3 treated group (25 mg/kg/day) showed one minutelesion with microscope. Compound 3 treated group (10 mg/kg/day) showedtumor in the left lung (FIGS. 11 and 12).

EXPERIMENTAL EXAMPLE 5-2 Melanoma Model in Mice (C57BL/6)

6 week female C57BL/6 mice (provided from Asan Institute for LifeSciences Animal Lab., Seoul, Korea) were housed in specific pathogenfree unit.

B16F10 cells (2×10⁴) suspended in 100 μl of RPMI 1640 serum-free mediumwere intravenously injected via tail vein. One week before tumor cellinjection, the following 3 groups were treated.

1) RPMI control group

2) Dendritic cells (DC) (5×10⁵ cells/day)+tumor lysate treated group

3) Compound 3 (50 mg/kg/day) treated group.

In the case of DC+tumor lysate treated group, one week before melanomainjection, 5×10⁵ DC cells mixed with tumor lysate were injected to theabdominal cavity every 1 weeks. In the case of Compound 3 treated group,50 mg/kg/day of Compound 3 was treated to each mouse. One week beforemelanoma (B16F10) injection, Compound 3 (50 mg/kg/day) via P.O wascontinued 2 weeks on and 1 week off for 6 weeks. As a result, grossfindings at 4^(th) week control group observed multiple metastaticlesions in both lungs. Compound 3 treated group and DC+tumor lysatetreated group showed no evidence of disease in the lung (FIGS. 13, 14)and showed 90% survival rate in the observation for 6 weeks aftermelanoma (B16F10) injection (FIG. 15).

Based on the presumption that the anti-cancer effects of the Compound 3are attributed to the activation of T-cells by Compound 3, cytotoxicityof T-cells activated by Compound 3 to malignant melanoma cells wasinvestigated. As a result, when the ratio of T-cells activated byCompound 3 to melanoma cells was 100:1, cytotoxicity was 42% increased(FIG. 16).

EXPERIMENTAL EXAMPLE 6 Toxicity Test of Compound 3

Synthesized Compound 3 was dissolved in 5% ethanol solution which wasorally administered at the 0.1 ml/20 g dose. Control group was treated5% ethanol solution. IRC mice, housed in SPF facility were used as testanimals. The animals were fasted for one day before drug administration,and had free access to water and chow thereafter. Eight to ten ICR mice,25-35 g of weight, were grouped. The test agent was orally administeredonce at increasing doses ranging from 62.5 mg/kg, 125 mg/kg, 250 mg/kg,500 mg/kg, 1.0 g/kg, to 2.0 g/kg. From the day of administration,survival number and any abnormal signs were observed with the naked eyefor 14 days. LD₅₀ was calculated by the method of Lichfield-Wilcoxon (

—acute toxicity test, Realize Inc., Tokyo, 1988), and weight changeswere calculated by the following Mathematical Formula.Weight increment rate(%)={Weight of day 14−Weight of day 0}÷{Weight ofday 0}×100  [Mathematical Formula 2]

The results are shown in Table 4. No toxicity was seen with 62.5 mg/kg˜2g/kg of Compound 3. This observation indicated that LD₅₀ was over 2g/kg. Any abnormal sign as not observed with the naked eyes for 14 daysafter the administration. The weight of animals in treated groupincreased steadily so did in control animals. As shown in Table 5, nosignificant, treatment-specific weight change (gaining or losing) wasobserved.

TABLE 4 Death rate by the treatment o Compound 3 (%) Dosage of Compound3 (mg/kg) 0 62.5 125 250 500 1000 2000 Number of dead mice/ 0/10 1/8 0/81/8 2/9 0/9 0/9 Number of orally administered mice LD₅₀ 0 12.5 30 12.522.2 0 0

TABLE 5 Weight increase 14 days after the oral-administration ofCompound 3 Dosage of Compound 3 (mg/kg) 0 63 125 250 500 1000 2000Weight 14 days after 38.7 ± 1.1 34.3 ± 0.1 38.1 ± 1.3 35.6 ± 1.4 36.5 ±1.9 35.5 ± 1.1 35.9 ± 2.7 Administration (g) Weight increment rate (%)16.9 11.9 17.3 11.2 10.1 9.5 14.4

Long term hepatotoxicity test was done on rats with Compound 3 dose at100 mg/Kg body weight/day given P.O. for 4 weeks, and liver functiontest, lipid profile, cytochrome C-450 activity were observed. At the endof 4 weeks liver histology was observed. No significantly adverse effectwas observed.

EXPERIMENTAL EXAMPLE 7 CLP (Cecal Ligation and Puncture) Test

CLP test was performed in order to confirm the effect of Compound 3 forprevention and treatment against septic shock. Ten 7-10 week old maleinbred C3H/HeN mice (20-25 g of weight) were grouped. After 50 mg/kg/dayof Compound 3 was orally administered to mice for 2 weeks on and 1 weekoff, mice were anesthetized with 80 mg/kg of ketamine and 16 mg/kg ofrompun. Septic shock was induced by CLP model in anesthetized mice. Onehour after inducing septic shock, 50 mg/kg of Compound 3 was treated,and then, the same treatment was continued for 3 days every 24 hours.Control group was orally administered PBS+5% ethanol solution. Survivalrate of Compound 3 treated group and control group with time lapse wasshown in Table 6. Compound 3 treated groups had 100% survival rate evenafter lapse of 120 hours.

TABLE 6 Survival rate in septic shock Survival rate with time lapse 0hour 24 hours 48 hours 72 hours 96 hours 120 hours Survival SurvivalSurvival Survival Survival Survival rate rate rate rate rate rate PBStreated group (control) 100% 60% 40% 40% 40% 40% Compound 3 (50 mg/kg)100% 100% 100% 100% 100% 100%

MANUFACTURING EXAMPLE 1 Preparation of Medical Supplies ContainingCompound 3 as an Effective Ingredient

After confirming through the above experiments that Compound 3 had anexcellent immunomodulating and anti-cancer activity, the presentinventors prepared a treatment containing Compound 3 as an effectiveingredient. Further, the followed manufacturing example of the treatmentcontaining Compound 3 as an effective ingredient can be applied not onlyto the preparing of treatment but also to the preparing of health food.If there isn't extra mention, the symbol of % means weight % in thefollowing manufacturing example.

MANUFACTURING EXAMPLE 1-1 Preparation of Soft Gelatin CapsulesMANUFACTURING EXAMPLE 1-1-1

Compound 3 30% Vitamin C 4.5%  Vitamin D3 0.001%   Manganese sulfate0.1%  Wax 10% Palm oil 25% Safflower oil (Carthamus tinctorius)30.399%   

MANUFACTURING EXAMPLE 1-1-2

Compound 3 31.25%  Evening primrose seed oil 59.75%  Soy oil 6.7%Vitamin E acetate ester (DL-α-tocopherol acetate) 2.1% Soy lecithin 0.2%

MANUFACTURING EXAMPLE 1-1-3

Compound 3 98.0% Vitamin E acetate ester (DL-α-tocopherol acetate) 2.0%

MANUFACTURING EXAMPLE 1-2 Preparation of Tablets

Compound 3 30% Vitamin C 10% Vitamin D3 0.001%   Manganese sulfate 0.1% Crystalline cellulose 25.0%   Lactose 32.999%    Magnesium Stearate  2%

MANUFACTURING EXAMPLE 1-3 Preparing of an Injection Formulation

Compound 3 2% Propylene glycol 35% Mono glyceride 8% Ethanol 5% Water50%

The injection formulation was prepared by the conventional method withabove mentioned compositions and contents.

MANUFACTURING EXAMPLE 2 Preparation of Medical Supplies ContainingCompound 1, 2, 4, and 5 as an Effective Ingredient

Soft gelatin capsules, tablets and injection suspension were prepared bythe same method and composition as described in the above manufacturingexample 1, except the Compound 3 was substituted with Compound 1, 2, 4,and 5 at the same ratio.

MANUFACTURING EXAMPLE 3 Preparation of Health Food Containing Compound 3as an Effective Ingredient

After confirming through the above examples that the Compound 3 had anexcellent immunomodulating, anti-septic shock, and anti-cancer activity,the present inventors prepared health food containing the same as aneffective ingredient.

MANUFACTURING EXAMPLE 3-1 Preparation of Beverages

Honey 522 mg Thioctic amide 5 mg Nicotinic amide 10 mg Sodium riboflavinhydrochloride 3 mg pyridoxine hydrochloride 2 mg Inositol 30 mg Orthoacid 50 mg Compound 3 0.48~1.28 mg water 200 ml

Beverage was prepared based on the above compositions and contents byfollowing a conventional method.

MANUFACTURING EXAMPLE 3-2 Preparation of Chewing Gum

Gum base 20%  Sugar 76.36~76.76% Compound 3 0.24~0.64% Fruit flavor 1%Water 2%

Chewing gum was prepared based on the above compositions and contents byfollowing a conventional method.

MANUFACTURING EXAMPLE 3-3 Preparation of Candy

Sugar 50~60% Starch syrup 39.26~49.66% Compound 3 0.24~0.64% Orangeflavor 0.1%

Candy was prepared based on the above compositions and contents byfollowing a conventional method.

MANUFACTURING EXAMPLE 3-4 Preparation of Biscuit

Strong flour 1^(st) class 88 kg Cake flour 1^(st) class 76.4 kg Refinedsugar 16.5 kg Salt 2.5 kg Glucose 2.7 kg Palm shortening 40.5 kg Ammo5.3 kg Baking soda 0.6 kg Sodium bisulfate 0.55 kg Rice flour 5.0 kgVitamin B1 0.003 kg Vitamin B2 0.003 kg Milk flavor 0.16 kg Water 71.1kg Whole milk powder 4 kg Substitute milk powder 1 kg Calcium phosphate,monobasic 0.1 kg Spraying salt 1 kg Spraying milk 25 kg Compound 30.2~0.5 kg

Biscuit was prepared based on the above compositions and contents byfollowing a conventional method.

MANUFACTURING EXAMPLE 3-5 Preparation of Ice Cream

Milk fat 10.0% Milk solids non-fat 10.8% Sugar 12.0% Starch syrup  3.0%Emulsifying stabilizer (span)  0.5% Flavor (Strawberry) 0.15% Water63.31~62.91% Compound 3 0.24~0.64%

Ice cream was prepared based on the above compositions and contents byfollowing a conventional method.

MANUFACTURING EXAMPLE 3-6 Preparation of Chocolate

Sugar 34.36~34.76% Cocoa butter 34% Cocoa mat 15% Cocoa powder 15%Lecithin 0.5%  Vanilla flavor 0.5%  Compound 3 0.24~0.64%

Chocolate was prepared based on the above compositions and contents byfollowing a conventional method.

MANUFACTURING EXAMPLE 4 Preparation of Health Food Containing Compound1, 2, 4, and 5 as an Effective Ingredient

Beverage, chewing gum, candy, biscuit, ice cream and chocolate wereprepared by the same method and composition as described in the abovemanufacturing example 3, except the Compound 3 was substituted withCompound 1, 2, 4, and 5 at the same ratio.

ADVANTAGEOUS EFFECTS

As explained hereinbefore, the mono acetyl diacyl glycerol derivativescontaining Compound 3 shows significant effect for immuno modulationincluding immune enhancing. In the case of inducing cancer in a hamsterby injecting cancer cell line, cancer development was delayed byactivating lymphocytes, monocytes, and dendritic cells that areimportant factors to promote immunity and apoptosis of cancer cell wasinduced by promoting cytotoxicity of immune cell against caner cell.Also in the case of mouse induced septic shock, it shows 100% survivalrate even after lapse of 120 hours by control of immune function andsuppression effect of apoptosis. Therefore, mono acetyl diacyl glycerolderivatives according to the present invention can be effectively usedfor an immunomodulating agent, a sepsis treatment, a cancer treatment,and a health food for an immune modulation or the prevention of cancer.

1. A method of treating sepsis comprising administering to a mammal aneffective amount of mono acetyl diacyl glycerol defined by formula II inan isolated form:


2. The method according to claim 1, wherein the mono acetyl diacylglycerol derivative is administered in a pharmaceutical composition inan amount of 20 to 100 wt %.
 3. The method according to claim 1, whereinthe mono acetyl diacyl glycerol derivative is administered in healthfood.